Embryonic stem cells capable of differentiating into desired cell lines

ABSTRACT

An embryonic stem cell which may be induced to differentiate homogeneously into a desired primary cell line. The embryonic stem cell may be engineered with DNA, which encodes a protein or polypeptide which promotes differentiation of the stem cell into a specific cell line, such as, for example, a neuronal cell line, a muscle cell line, or a hematopoietic cell line. The DNA may encode a transcription factor found in the particular cell line. In another alternative, a desired cell line is produced from embryonic stem cells by culturing embryonic stem cells under conditions which provide for a three-dimensional network of embryonic stem cells, and then stimulating embryonic stem cells with an agent, such as retinoic acid, or dimethylsulfoxide, which promotes differentiation of the embryonic stem cells into the desired cell line, such as, for example, a neuronal cell line, or a muscle cell line.

[0001] This application is a continuation-in-part of application Ser.No. 08/145,175, filed Nov. 3, 1993.

[0002] This invention relates to embryonic stem cells. Moreparticularly, this invention relates to embryonic stem cells which areengineered with DNA and/or cultured in the presence of an agent, wherebysuch cells become capable of differentiating homogeneously into adesired primary cell line. Such homogeneous differentiation has not andcannot be achieved unless the methods described herein are applied.

[0003] Embryonic stem cells are pluripotent cells derived from the innercell mass of pre-implantation embryos. (Evans et al., Nature, Vol. 292,pgs. 154-156 (1981)). Embryonic stem cells can differentiate into anycell type in vivo (Bradley, et al.,Nature, Vol. 309, pgs. 255-256(1984); Nagy, et al., Development, Vol. 110, pgs. 815-821 (1990) andinto a more limited variety of cells in vitro (Doetschman, et al., J.Embryol. Exp. Morph., Vol. 87, pgs. 27-45 (1985); Wobus, et al., Biomed.Biochim. Acta, Vol. 47, pgs. 965-973 (1988); Robbins, et al., J. Biol.Chem., Vol. 265, pgs. 11905-11909 (1990); Schmitt, et al., Genes andDevelopment, Vol. 5, pgs. 728-740 (1991)). Embryonic stem cells,however, are more difficult to maintain in the laboratory and requirethe addition of a differentiation inhibitory factor (commonly referredto as leukemia inhibitory factor (or LIF) in the culture medium toprevent spontaneous differentiation (Williams, et al., Nature, Vol. 336,pgs. 684-687 (1988); Smith, et al., Nature, Vol. 336, pgs. 688-690(1988); Gearing, et al., Biotechnology, Vol. 7, pgs. 1157-1161 (1989);Pease, et al., Dev. Biol., Vol. 141, pgs. 344-352 (1990). LIF is asecreted protein and can be provided by maintaining embryonic stem cellson a feeder layer of cells that produce LIF (Evans, et al., 1981;Robertson, Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, Washington, D.C.: IRL Press (1987)) or by the addition ofpurified LIF (Williams, et al., 1988; Smith, et al., 1988; Gearing, etal., 1989; Pease, et al., Exp. Cell Res., Vol. 190, pgs. 209-211 (1990)to the medium in the absence of feeder layers. Differentiation ofembryonic stem cells into a heterogeneous mixture of cells occursspontaneously if LIF is removed, and can be induced further bymanipulation of culture conditions (Doetschmann, et al., 1985; Wobus, etal., 1988; Robbins, et al., 1990; Schmitt, et al., 1991; Wiles, et al.,Development, Vol. 111, pgs. 254-267 (1991); Gutierrez-Ramos, et al.,Proc. Nat. Acad. Scil., Vol. 89, pgs. 9111-9175 (1992)). Differentiationof stem cells into a homogeneous population, however, has not beenachieved. Embryonic stem cell differentiation can be variable betweendifferent established embryonic stem cell lines and even betweenlaboratories using the same embryonic stem cell lines.

[0004] It is an object of the present invention to provide embryonicstem cells which are capable of differentiating uniformly into aspecific and homogeneous cell line, not achievable by previous methods.

[0005] In accordance with an aspect of the present invention, there isprovided a method of producing a desired cell line from embryonic stemcells. The method comprises culturing embryonic stem cells underconditions which promote growth of the embryonic stem cells at anoptimal growth rate. The embryonic stem cells then are cultured underconditions which promote the growth of the cells at a rate which is lessthan that of the optimal growth rate, and in the presence of an agentwhich promotes differentiation of the embryonic stem cells into thedesired cell line.

[0006] In general, a growth rate which is less than the optimal growthrate, is a growth rate from about 10% to about 80%, preferably fromabout 20% to about 50%, of the maximum growth rate for embryonic stemcells. The growth rates for embryonic stem cells can be determined fromthe doubling times of the embryonic stem cells. In general, the optimumdoubling time for embryonic stem cells is from about 13 hours to about18 hours, and more particularly, from about 15 hours to about 16 hours.

[0007] In one embodiment, when the embryonic cells are being culturedunder conditions which promote growth of the cells at an optimal growthrate, the embryonic stem cells are cultured in the presence of a mediumincluding leukemia inhibitory factor (LIF), and serum selected from thegroup consisting of: (i) horse serum at a concentration of from about 5%by volume to about 30% by volume; and (ii) fetal bovine serum at aconcentration of from about 15% by volume to about 30% by volume. In oneembodiment, the serum is horse serum at a concentration of about 10% byvolume. In another embodiment, the serum is fetal bovine serum at aconcentration of about 15% by volume.

[0008] In yet another embodiment, when the embryonic stem cells arecultured at an optimal growth rate, the embryonic stem cells arecultured in the absence of a feeder layer of cells.

[0009] In one embodiment, the agent which promotes differentiation ofthe embryonic stem cells is selected from the group consisting ofretinoic acid and nerve growth factor, and the desired cell line is aneuronal cell line.

[0010] In one embodiment, in addition to culturing the cells in thepresence of the stimulating agent selected from the group consisting ofretinoic acid and nerve growth factor, the embryonic stem cells aregrown in the presence of a cytokine. Cytokines which may be employedinclude, but are not limited to, Interleukin-1, Interleukin-3,Interleukin-4, Interleukin-6, colony stimulating factors such as M-CSF,GM-CSF, and CSF-1, steel factor, and erythropoietin.

[0011] In a further embodiment, the agent which promotes differentiationof the embryonic stem cells is selected from the group consisting ofdimethylsulfoxide and hexamethylene bis-acrylamide, and the desired cellline is a muscle cell line, such as a smooth muscle cell line, or askeletal muscle cell line, or a cardiac muscle cell line. In oneembodiment, the agent is dimethylsulfoxide. In another embodiment, theagent is hexamethylene bis-acrylamide.

[0012] In one embodiment, in addition to culturing the embryonic stemcells in the presence of an agent which promotes differentiation of theembryonic stem cells into a muscle cell line, the embryonic stem cellsalso are grown in the presence of a cytokine, examples of which arehereinabove described.

[0013] In yet another embodiment, when the embryonic stem cells arecultured in the presence of the agent which promotes differentiation ofthe embryonic stem cells into a desired cell line, the embryonic stemcells also are cultured in the presence of fetal bovine serum at aconcentration of about 10% by volume.

[0014] In a further embodiment, when the embryonic stem cells arecultured in the presence of the agent which promotes differentiation ofthe embryonic cells into a desired cell line, the embryonic stem cellsalso are cultured in a three-dimensional format.

[0015] Thus, Applicants have found that one may produce a homogenousdesired cell line from embryonic stem cells by culturing the embryonicstem cells initially under conditions which favor the growth orproliferation of such embryonic stem cells at an optimal growth rate,and then culturing the cells under conditions which decrease the growthrate of the cells and promote differentiation of the cells to a desiredcell type.

[0016] In a preferred embodiment, the embryonic stem cells cultured in astandard culture medium (such as, for example, Minimal EssentialMedium), which may include supplements such as, for example, glutamine,and β-mercaptoethanol. The medium may also include leukemia inhibitoryfactor (LIF), or factors with LIF activity, such as, for example, CNTFor IL-6, and horse serum. LIF, and factors with LIF activity, preventsspontaneous differentiation of the embryonic stem cells, and is removedprior to the addition of the agent. Horse serum promotes differentiationof the embryonic stem cells into the specific cell type after theaddition of the agent to the medium. After the cells have been culturedfor a period of time sufficient to permit the cells to proliferate to adesired number, the cells are washed free of LIF, and then culturedunder conditions which provide for the growth of the cells at adecreased growth rate but which also promote differentiation of thecells. The cells are cultured in the presence of an agent which promotesor stimulates differentiation of the embryonic stem cells into a desiredcell line, and in the presence of fetal bovine serum at a concentrationof from about 5% by volume to about 10% by volume, preferably at about10% by volume. The presence of the fetal bovine serum at a concentrationof from about 5% by volume to about 10% by volume, and of the agent,provides for growth or proliferation of the cells at a rate which isless than the optimal rate, while favoring the differentiation of thecells into a homogeneous desired cell type. The desired cell type isdependent upon the agent which promotes or stimulates thedifferentiation of the embryonic stem cells. The embryonic stem cellsalso are cultured in a three-dimensional format. Examples of suchthree-dimensional culturing formats are disclosed in Doetschman, et al.,(1985), and in Rudnicki, et al., (1987).

[0017] For example, the embryonic stem cells may be placed in a culturevessel to which the cells do not adhere. Examples of non-adherentsubstrates include, but are not limited to, polystyrene and glass. Thesubstrate may be untreated, or may be treated such that a negativecharge is imparted to the cell culture surface. In addition, the cellsmay be plated in methylcellulose in culture media, or in normal culturemedia in hanging drops (Rudnicki, et al., 1987). Media which containsmethylcellulose is viscous, and the embryonic stem cells cannot adhereto the dish. Instead, the cells remain isolated, and proliferate, andform aggregates.

[0018] In order to form aggregates in hanging drops of media, cellssuspended in media are spotted onto the underside of a lid of a culturedish, and the lid then is placed on the culture vessel. The cells, dueto gravity, collect on the undersurface of the drop and form aggregates.

[0019] In accordance with another aspect of the present invention, thereis provided an embryonic stem cell. The embryonic stem cell has beenengineered with DNA which encodes a protein or polypeptide whichpromotes differentiation of the cell into a specific cell line.

[0020] The DNA which encodes a protein or polypeptide which promotesdifferentiation of the embryonic stem cell into a specific cell line isDNA encoding a protein or polypeptide which is normally found in thespecific differentiated cell line. Preferably, the protein orpolypeptide which is present in the specific cell line is a protein orpolypeptide which generally is not present in other types of cells.

[0021] In one embodiment, the DNA which encodes a protein or polypeptidewhich promotes differentiation of the embryonic stem cell into aspecific differentiated cell line is DNA encoding a transcription factorpresent in the specific cell line to promote differentiation of the cellinto the specific cell line.

[0022] In one embodiment, the DNA encoding a transcription factor is DNAencoding a transcription factor present in neuronal cells, and thespecific cell line is a neuronal cell line.

[0023] In another embodiment, the DNA encoding a transcription factor isDNA encoding a transcription factor, such as the MyoD gene, present inmuscle cells, and the specific cell line is a muscle cell line.

[0024] In yet another embodiment, the DNA encoding a transcriptionfactor is DNA encoding a transcription factor present in hematopoieticcells, and the specific cell line is a hematopoietic cell line.

[0025] The DNA which encodes a protein or polypeptide which promotesdifferentiation of the embryonic cell into a specific cell line may beisolated in accordance with standard genetic engineering techniques(such as, for example, by isolating such DNA from a cDNA library of thespecific cell line) and placed into an appropriate expression vector,which then is transfected into embryonic stem cells.

[0026] Appropriate expression vectors are those which may be employedfor transfecting DNA into eukaryotic cells. Such vectors include, butare not limited to, prokaryotic vectors such as, for example, bacterialvectors; eukaryotic vectors, such as, for example, yeast vectors andfungal vectors; and viral vectors, such as, but not limited to,adenoviral vectors, adeno-associated viral vectors, and retroviralvectors. Examples of retroviral vectors which may be employed include,but are not limited to, those derived from Moloney Murine LeukemiaVirus, Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus.

[0027] In a preferred embodiment, cDNA is synthesized from RNA isolatedby the method of Chomczynski, et al., Anal. Biochem., Vol. 162, pgs.156-159 (1987) from cells of interest. All RNA preparations are screenedfor the presence of large RNAs with gene probes that recognize highmolecular weight mRNA (i.e., greater than 6 kb) on Northern blots. Forexample, all RNA preparations from neural cells may be screened fordetection of MAP2 mRNA on Northern blots. (MAP2 is a brain specificprotein present in low abundance and coded for by a messenger RNA ofabout 9 kb. The ability to detect MAP2 messenger RNA on a Northern blotis a stringent test for the presence of intact high quality RNA.)

[0028] For cDNA synthesis, a single tube method developed by Gubler,Nucl. Acids Res., Vol. 16, pg. 2726 (1988) is employed, and conditionsare optimized to yield the greatest amount of full length cDNA product(about 7.5 kb in length). The cDNA is inserted into the pcDNA3 vector(Invitrogen), which allows for expression of the cDNA insert inmammalian cells. The pcDNA3 vector contains the cytomegalovirus (CMV)promoter, the SV40 origin of replication, the neomycin resistance genefor selection in eukaryotic cells, and the ampicillin resistance genefor selection in bacteria such as E.coli.

[0029] cDNA libraries are constructed wherein all the clones areoriented in the proper orientation for expression. Such is achieved bysynthesizing oligo (dT) primed libraries with an oligo (dT) primer thatincludes a NotI site, and after cDNA synthesis, a BstXI adapter isligated to the cDNA. Finally, the cDNA is digested with NotI (an enzymethat cuts infrequently in eukaryotic genes), thus creating a cDNA with aNotI overhang at the 3′ end and a BstXI overhang at the 5′ end. The cDNAthen is ligated into pcDNA3 digested with BstXI and NotI. This placesthe 5′ end of the cDNA downstream from the CMV promoter.

[0030] To enrich for developmentally expressed genes, libraries fromuninduced embryonic stem cells are screened with labeled cDNA fromdifferentiated embryonic stem cells and all cross-hybridizing clones areeliminated from further analysis. Such method allows the removal ofthose elements common to differentiated and undifferentiated cells.Also, subtractive cDNA libraries are constructed according to the methodof Sive, et als., Nucl. Acids Res., Vol. 16, pg. 10937 (1988).Subtractive cDNA libraries are cDNA libraries that are enriched forgenes expressed in one cell type but not in another. The method relieson removal of common DNA sequences through hybridization of similar DNAsequences, and then the removal of these hybridized double-strandedDNAs. A subtractive cDNA library that contains sequences specific for aparticular cell type derived from induced embryonic stem cells isgenerated. Single stranded cDNA is synthesized from uninduced cells. Toselect for those genes that are specific for the desired cell linederived from embryonic stem cells, genes that are expressed both in theinduced cells and the non-induced embryonic stem cells are removed.Thus, RNA which is isolated and purified from embryonic stem cells thathave differentiated into a desired cell line is hybridized to an excessof cDNA synthesized from uninduced embryonic stem cells to insure thatall common elements are removed. RNA and cDNA common to both the inducedand uninduced embryonic stem cells will hybridize, and these hybrids areremoved. To remove double-stranded material, cDNA from uninducedembryonic stem cells is covalently modified with photoactivatable biotin(Sive, et al., 1988), and the hybrid can be removed by a simple phenolextraction because the biotin on the cDNA will cause the hybrid topartition to the phenol phase while the non-hybridized RNA willpartition to the aqueous phase. Following this selection, RNA speciesfound specifically in differentiated embryonic stem cells are used toconstruct cDNA libraries as hereinabove described.

[0031] Plasmid DNA containing cDNA inserts then are electroporated intoembryonic stem cells. Cells are transfected with a plasmid that containssequences for neomycin resistance and stable transfectants are isolatedbased on neomycin resistance. Stable transfected clones are isolated andinduced with an appropriate agent, or with leukemia inhibitory factor(LIF) withdrawal alone, and scored for an increased ability todifferentiate in response to these induction signals. Clones also areexamined to determine if they are differentiating spontaneously in thepresence of LIF.

[0032] In accordance with another aspect of the present invention, thereis provided a method of producing a desired cell line from embryonicstem cells. The method comprises engineering embryonic stem cells withDNA which encodes a protein or polypeptide which promotesdifferentiation of the embryonic stem cells into a specific cell line.The embryonic stem cells then are stimulated with an agent whichpromotes differentiation of the embryonic stem cells into the desiredcell line.

[0033] In one embodiment, the DNA which encodes a protein or polypeptidewhich promotes differentiation of the embryonic stem cells into aspecific cell line is DNA encoding a transcription factor present inneuronal cells and said agent is selected from the group consisting ofretinoic acid and nerve growth factor. In one alternative, the cellsalso may be grown in the presence of a cytokine such as thosehereinabove described.

[0034] In another embodiment, the DNA which encodes a protein orpolypeptide which promotes differentiation of the embryonic stem cellsinto a specific cell line is DNA encoding a transcription factor, suchas, for example, the MyoD gene, present in muscle cells and said agentis a bipolar agent such as dimethylsulfoxide or hexamethylenebis-acrylamide. In one alternative, the embryonic stem cells also may begrown in the presence of a cytokine.

[0035] The embryonic stem cells may be engineered with the DNA andcultured under conditions hereinabove described. For example, prior toinduction, the embryonic stem cells are engineered with DNA whichencodes a protein or polypeptide which promotes differentiation of theembryonic stem cells into a specific cell line. Then, the embryonic stemcells may be cultured under conditions which provide for athree-dimensional network of such cells.

[0036] Also, it is to be understood that, within the scope of thepresent invention, that the embryonic stem cells may be used for genetherapy purposes. The embryonic stem cells may be engineered with DNAencoding a desired therapeutic agent. Such engineering may beaccomplished by using expression vectors such as those hereinabovedescribed. Once the cells are engineered with DNA encoding a desiredtherapeutic agent, the cells then are engineered with DNA which encodesa protein or polypeptide which promotes differentiation of the embryonicstem cells into a specific desired cell line, and/or stimulated with anagent which promotes differentiation of the embryonic stem cells into adesired cell line. The differentiated cells then may be administered toa host, such as a human or non-human host, as part of a gene therapyprocedure.

[0037] In addition, there is also provided within the scope of thepresent invention, a method of screening embryonic stem cells forproteins which induce differentiation of embryonic stem cells intodesired cell lines. In such method, RNA is obtained from specificallydesired cells or tissues (such as for example, brain cells), and cDNAlibraries are then constructed and placed into expression vectors. Thelibraries may be normal cDNA libraries or they may be subtractive cDNAlibraries, i.e., such DNA libraries include DNA found in the desiredcells or tissues but not in other cells or tissues. The expressionvectors are then transfected into eukaryotic cells, such as COS cells.The cell culture supernatant then may be applied to embryonic stem cellcultures to determine if any secreted proteins from such cells inducedifferentiation of embryonic stem cells to a specific cell type. ThecDNA from cells which induce differentiation of embryonic stem cells toa specific cell type then is evaluated further in order to determinewhich individual clones of such cDNA libraries induce differentiation ofembryonic stem cells to a specific cell type. Once a specific cDNA whichinduces differentiation of embryonic stem cells to a desired cell typeis identified, such cDNA then may be isolated and cloned into anappropriate expression vector, which may be transfected intoundifferentiated embryonic stem cells or the expressed, purified proteinmay be added directly to cultured embryonic stem cells.

[0038] In one embodiment, such screening may be carried out by poolingbacterial clones, from the cDNA library prepared as hereinabovedescribed, into groups of 1,000, and isolating plasmid DNA from thepooled clones. The plasmid DNA's then are electroporated into COS cells,such as COS-7 cells, for expression. After allowing from 48 to 72 hoursfor expression of transfected genes, tissue culture supernatant fromtransfected COS cells is harvested and applied to embryonic stem cellsto determine if any secreted proteins from the COS cells can inducedifferentiation of embryonic stem cells. Supernatants from mocktransfected cells (cells transfected with the plasmid alone) are testedin parallel to control for any non-specific effects of COS cell derivedproteins. Embryonic stem cell differentiation may be screened by severalmeans: (i) by microscopic observation of overt changes in embryonic stemcell morphology; (ii) by measuring changes in neuron specific geneexpression on Northern blots with probes to neuron specific markers suchas neuron specific enolase, GAP-43, and MAP2; and (iii) by loss ofexpression of a carbohydrate surface marker present only onundifferentiated stem cells recognized by the monoclonal antibody SSEA-1(Ozawa, et al., Cell. Diff., Vol. 16, pp. 169-173 (1985)).

[0039] When a pool has been identified that expresses inducing capacity,that pool of cDNA clones is broken down further into smaller pools of100 clones, and these sub-pools are transfected into COS cells.Supernatants are screened for inducing activity. Once appropriatesub-pools are identified, the clones are plated in 96 well dishes, androws and columns are combined. The pooled columns and rows then aretransfected into COS cells, and supernatants again are screened foractivity. By analyzing the columns and rows that exhibit activity, theexact clone expressing inducing activity can be identified. This clonethen is tested for ability to induce differentiation. After initialidentification of potential factors, full-length cDNA clones areisolated and sequenced. Sequenced clones then are compared to othercloned genes in the DNA data base for homology or identity withpreviously cloned genes. Once a novel gene is identified, the gene iscloned into a stable expression system, the protein is purified, and itsbiological activity is tested. Sequencing of DNA is performed bystandard protocols. Biologically active protein is prepared by standardchromatographic methods.

[0040] Alternatively, cDNA from differentiating embryonic stem cells orfrom embryonic organs and brain regions can be introduced directly intoembryonic stem cells, and embryonic stem cell supernatants are screenedfor inducing activity.

[0041] The differentiated stem cells may be employed by means known tothose skilled in the art to treat a variety of diseases or injuries. Forexample, stem cells which have differentiated into neuronal cells may beadministered to a patient, such as, for example, by transplanting suchcells into a patient, to treat diseases such as Huntington's disease,Parkinson's disease, and Alzheimer's disease. Such neuronal cells alsomay be employed to treat spinal cord injuries or chronic pain. Also,stem cells which have differentiated into muscle cells may be employedin treating muscular dystrophy, cardiomyopathy, congestive heartfailure, and myocardial infarction, for example.

[0042] The invention will now be described with respect to the followingexamples; however, the scope of the present invention is not intended tobe limited thereby.

EXAMPLE 1

[0043] Undifferentiated embryonic stem cells (ES-E14TG2a, purchased fromthe American Type Culture Collection, catalog no. ATCC CRL 1821) aremaintained in Dulbecco's modified Minimal Essential Medium (DMEM)supplemented with glutamine, β-mercaptoethanol, 10% (by volume) horseserum, and human recombinant Leukemia Inhibitory Factor (LIF). The LIFreplaces the need for maintaining embryonic stem cells on feeder layersof cells, and is essential for maintaining embryonic stem cells in anundifferentiated state.

[0044] In order to promote the differentiation of the embryonic stemcells into neuronal cells, the embryonic stem cells are trypsinized andwashed free of LIF, and placed in DMEM supplemented with 10% (by volume)fetal bovine serum (FBS). After resuspension in DMEM and 10% FBS, 1×10⁶cells are plated in 5 ml DMEM plus 10% FBS plus 0.5 μM retinoic acid ina 60 mm Fisher brand bacteriological grade Petri dish. In such Petridishes, embryonic stem cells cannot adhere to the dish, and insteadadhere to each other, thus forming small aggregates of cells.Aggregation of cells aids in enabling proper cell differentiation. Aftertwo days, aggregates of cells are collected and resuspended in freshDMEM plus 10% FBS plus 0.5 μM retinoic acid, and replated in Petridishes for an additional two days. Aggregates, now induced four dayswith retinoic acid, are trypsinized to form a single-cell suspension,and plated in serum-free medium on poly-D-lysine-coated tissue culturegrade dishes. The stem cell medium is formulated with Kaighn's modifiedHam's F12 as the basal medium with the following supplements added:

[0045] 15 μg/ml ascorbic acid

[0046] 0.25% (by volume) calf serum

[0047] 6.25 μg/ml insulin

[0048] 6.25 μg/ml transferrin

[0049] 6.25 μg/ml selenous acid

[0050] 5.35 μg/ml linoleic acid

[0051] 30 pg/ml thyroxine (T3)

[0052] 3.7 ng/ml hydrocortisone

[0053] 1 ng/ml Heparin 10 ng/ml somatostatin

[0054] 10 ng/ml Gly-His-Lys (liver cell growth factor)

[0055] 0.1 μg/ml epidermal growth factor (EGF)

[0056] 50 μg/ml bovine pituitary extract (BPE)

[0057] Such medium provides for consistent differentiation of the stemcells into neuronal cells, and provides for survival of the neuronalcells for a period of time greater than 3 days, and selectively removesdividing non-neuronal cells from the population. The poly-D-lysinepromotes the attachment of the neuronal cells to the tissue cultureplastic, and prevents detachment of the cells from the dish and theforming of floating aggregates of cells. The cells are cultured for 5days. Upon culturing of the cells in the above medium, a culture ofcells in which greater than 90% of the cells are neuronal cells isobtained. Such neuronal cells, which express the neurotransmitter gammaamino butyric acid (GABA), then may be employed for the treatment of theneural degeneration disease Huntington's disease. Through geneticengineering, these cells can be directed to express dopamine (for thetreatment of Parkinson's disease) or acetylcholine (for the treatment ofAlzheimer's disease).

EXAMPLE 2

[0058] Undifferentiated embryonic stem cells (ES-D3, purchased from theAmerican Type Culture Collection as ATCC catalog no. ATCC CRL 1934) aremaintained in supplemented Dulbecco's modified Minimal Essential Mediumas described in Example 1. The embryonic stem cells then are trypsinizedand washed free of LIF and placed in 1% (by volume) dimethylsulfoxide inDMEM plus 10% horse serum. Two days after the addition ofdimethylsulfoxide and plating of cells in Petri dishes to formaggregates, the aggregates are collected and resuspended in fresh mediumplus 1% dimethylsulfoxide. The aggregates are then plated ontomulti-well untreated culture grade dishes without trypsin treatment. Oneaggregate is plated per well. The aggregates are cultured for 5 days.Upon culturing of the cells in multi-well dishes, cell cultures in whichgreater than 80% of the aggregates contain contracting muscle cells areobtained. Such cells may be used to treat cardiomyopathies, myocardialinfarction, congestive heart failure, or muscular dystrophy.

EXAMPLE 3 Transfection of Embryonic Stem Cells with Mouse MyoD cDNA

[0059] For transfection of embryonic stem cells with mouse MyoD cDNA,both the D3 (ATCC catalog no. CRL 1934) and E14 TG2a (ATCC catalog no.CRL 1821) embryonic stem cell lines were used. Embryonic stem cells werecultured as described in Robertson, 1987, except that the cells weremaintained in media containing 5 ng/ml human recombinant leukemiainhibitory factor instead of on feeder layers.

[0060] Embryonic stem cells were co-transfected with pKJ1-Neo (Dinsmore,et al., Cell, Vol. 64, pgs. 817-826 (1991)), which carries the neomycinresistance gene for selection of stable transfectants, and with pEMCII(Davis, et al., Cell, Vol. 51, pgs. 987-1000 (1987)), which contains aportion of the mouse MyoD cDNA. pKJ1-Neo was linearized at the uniqueNsiI site and pEMCII was linearized at the unique ScaI site. In order tointroduce the linearized plasmids into the embryonic stem cells, theembryonic stem cells were electroporated using a Gene Pulser (Bio Rad)in 0.4 cm gap distance electroporation cuvettes with the Gene Pulser setat 240 volts, 500μ Farads. For electroporation, 8×10⁶ embryonic stemcells were suspended in 1 ml of HEPES-buffered saline (25 mM HEPES, 134mM Na Cl, 5 mM KCl, 0.7 mM Na₂HPO₄, pH 7.1) with 2 μg of linearizedpKJ1-Neo and 20 μg pEMCII. After electroporation, the cells were platedat 5-7×10⁵ per 35 mm gelatin coated culture dish in growth mediumcontaining recombinant human leukemia inhibitory factor. The cells wereallowed to grow for 36 hours and then Geneticin (Gibco-BRL), acommercial brand of neomycin, was added to the medium at a concentrationof 400 μg/ml. The media containing the Geneticin was changed daily untilclones of neomycin resistant cells could be identified (7 days afterGeneticin addition). Individual neomycin resistant clones were isolatedusing glass cloning cylinders (Bellco).

[0061] Stable transfectants were isolated, expanded, frozen, and thenstored in liquid nitrogen. 35 independent stably transfected embryonicstem cell lines were isolated. Ten of these cell lines have beenanalyzed, and have been found to express different amounts of MyoD asdetected by Northern blots. Embryonic stem cell lines that were found toexpress high levels of MyoD RNA were found to have embryonic stem cellsin the population that spontaneously differentiated into muscle cells asassessed by the staining of cells with a muscle specific myosinantibody. Those cell lines which showed high levels of MyoD expressionwere characterized further by inducing differentiation withdimethylsulfoxide. Cell lines which expressed high amounts of MyoDdifferentiated almost exclusively into skeletal muscle afterdimethylsulfoxide induction. The percentage of cells that differentiatedinto skeletal muscle was greater than 90% as assessed by staining formuscle specific myosin, and by the ability of these cells to fuse andform myotubes that spontaneously twitch. In contrast, MyoD transformantsthat expressed very low amounts of MyoD differentiated into a mix ofcardiac, smooth, and skeletal muscle indistinguishable from that derivedfrom non-transfected embryonic stem cells. Additionally, there was nodetectable difference between the D3 and E14 embryonic stem cell linesfor MyoD expression or differentiation.

[0062] It is to be understood, however, that the scope of the presentinvention is not to be limited to the specific embodiments describedabove. The invention may be practiced other than as particularlydescribed and still be within the scope of the accompanying claims.

What is claimed is:
 1. A method of producing a desired cell line fromembryonic stem cells, comprising: (a) culturing embryonic stem cellsunder conditions which promote growth of said cells at an optimal growthrate; and (b) culturing said embryonic stem cells under conditions whichpromote growth of said cells at a rate which is less than that of saidoptimal growth rate, and in the presence of an agent which promotesdifferentiation of said embryonic stem cells into said desired cellline.
 2. The method of claim 1 wherein, in step (a), said embryonic stemcells are cultured in the presence of a medium including leukemiainhibitory factor, CNTF, or IL-6; and serum selected from the groupconsisting of (i) horse serum at a concentration of from about 5% byvolume to about 30% by volume; and (ii) fetal bovine serum at aconcentration of from about 15% by volume to about 30% by volume.
 3. Themethod of claim 2 wherein, in step (a), said serum is horse serum at aconcentration of about 10% by volume.
 4. The method of claim 2 wherein,in step (a), said serum is fetal bovine serum at a concentration ofabout 15% by volume.
 5. The method of claim 1 wherein, in step (a), saidembryonic stem cells are cultured in the absence of a feeder layer ofcells.
 6. The method of claim 1 wherein, in step (b), said agent isselected from the group consisting of retinoic acid and nerve growthfactor, and said desired cell line is a neuronal cell line.
 7. Themethod of claim 6 wherein said agent is retinoic acid.
 8. The method ofclaim 7 wherein, in addition to culturing said embryonic stem cells inthe presence of said retinoic acid, said embryonic stem cells are grownin the presence of a cytokine.
 9. The method of claim 1 wherein, in step(b), said embryonic stem cells are cultured in the presence of fetalbovine serum at a concentration of about 10% by volume.
 10. Neuronalcells produced by the method of claim
 6. 11. The method of claim 1wherein, in step (b), said agent is selected from the group consistingof dimethylsulfoxide and hexamethylene bis-acrylamide, and said desiredcell line is a muscle cell line.
 12. The method of claim 11 wherein saidagent is dimethylsulfoxide.
 13. The method of claim 11 wherein saidagent is hexamethylene bis-acrylamide.
 14. The method of claim 11wherein, in addition to culturing said embryonic stem cells in thepresence of an agent selected from the group consisting ofdimethylsulfoxide and hexamethylene bis-acrylamide, said embryonic stemcells are grown in the presence of a cytokine.
 15. Muscle cells producedby the method of claim
 11. 16. An embryonic stem cell, said embryonicstem cell having been engineered with DNA which encodes a protein orpolypeptide which promotes differentiation of said cell into a specificcell line.
 17. The cell of claim 16 wherein said DNA which encodes aprotein or polypeptide which promotes differentiation of said cell intoa specific cell line is DNA encoding a transcription factor present insaid specific cell line to promote differentiation of said cell intosaid specific cell line.
 18. The cell of claim 17 wherein said DNAencoding a transcription factor is DNA encoding a transcription factorpresent in neuronal cells, and said specific cell line is a neuronalcell line.
 19. The cell of claim 17 wherein said DNA encoding atranscription factor present in a specific cell line is DNA encoding atranscription factor present in muscle cells, and said specific cellline is a muscle cell line.
 20. The cell of claim 17 wherein said DNAencoding a transcription factor is DNA encoding a transcription factorpresent in hematopoietic cells, and the specific cell line is ahematopoietic cell line.
 21. Neuronal cells produced from the cell ofclaim
 18. 22. Muscle cells produced from the cell of claim
 19. 23.Hematopoietic cells produced from the cell of claim
 20. 24. A method ofproducing a desired cell line from embryonic stem cells, comprising:engineering said embryonic stem cells with DNA which encodes a proteinor polypeptide which promotes differentiation of said embryonic stemcells into a specific cell line; and stimulating said embryonic stemcells with an agent which promotes differentiation of said embryonicstem cells into said desired cell line.
 25. The method of claim 24wherein said DNA which encodes a protein or polypeptide which promotesdifferentiation of said embryonic stem cells into a specific cell lineis DNA encoding a transcription factor present in neuronal cells andsaid agent is retinoic acid.
 26. Neuronal cells produced by the methodof claim
 25. 27. The method of claim 24 wherein said DNA which encodes aprotein or polypeptide which promotes differentiation of said embryonicstem cells into a specific cell line is DNA encoding a transcriptionfactor present in muscle cells and said agent is selected from the groupconsisting of dimethylsulfoxide and hexamethylene bis-acrylamide. 28.Muscle cells produced by the method of claim
 27. 29. A method ofscreening embryonic stem cells for proteins which induce differentiationof embryonic stem cells into a desired cell line comprising: (a)obtaining RNA from a desired cell or tissue; (b) constructing cDNAlibraries from said RNA obtained from said desired cell or tissue; (c)cloning such cDNA libraries into expression vectors; (d) transfectingsaid vectors into eukaryotic cells; (e) contacting embryonic stem cellswith culture supernatant from said transfected eukaryotic cells todetermine if proteins contained in said culture supernatant inducedifferentiation of said embryonic stem cells into a desired cell line;(f) obtaining cDNA clones from those cells which induce differentiationof embryonic stem cells into a desired cell line; (g) transfecting saidcDNA clones from said cells which induce differentiation of embryonicstem cells into a desired cell line into eukaryotic cells; (h)contacting embryonic stem cells with culture supernatant obtained fromthe eukaryotic cells of step (g); (i) obtaining cDNA clones from thosecells which induce differentiation of embryonic stem cells into adesired cell line; (j) transfecting said cDNA clones from step (i) intoembryonic stem cells; and (k) determining at least one cDNA clone whichinduces differentiation of embryonic stem cells into a desired cellline.